Organic anion transporter 5 renal expression and urinary excretion in rats exposed to mercuric chloride: a potential biomarker of mercury-induced nephropathy

Mercuric chloride (HgCl₂) induces acute kidney injury (AKI) affecting glomerular hemodynamics and, more specifically, the pars recta (S3 segment) of the proximal tubule. The organic anion transporter 5 (Oat5) is exclusively localized in the apical membranes of S3 segment. Oat5 urinary excretion was recently proposed as potential early biomarker of ischemic AKI. The aim of this study was to evaluate the renal expression and the urinary excretion of the Oat5 in rats exposed to HgCl₂. Male Wistar rats were treated with a single injection of HgCl₂ at different doses of 0, 0.2, 1 and 5 mg/kg body wt (control, Hg0.2, Hg1 and Hg5 groups). The renal expression of Oat5 was evaluated by immunohistochemistry, Western blotting, and real-time PCR. Oat5 and sodium dicarboxylate cotransporter 1 (NaDC1) abundances and alkaline phosphatase activity (AP) were assayed in urine. An HgCl₂ dose-related decrease in Oat5 mRNA levels and in Oat5 protein levels in renal homogenates was observed. Hg5 rats showed an increase in urinary excretion of Oat5 and NaDC1 as well as alterations of other widely used parameters for renal dysfunction and injury (plasma creatinine, plasma urea, urinary AP activity, kidney weight, histological lesions). In Hg0.2 group only an increase of urinary excretion of Oat5 was observed. The increase of Oat5 urinary excretion in Hg1 group was associated to the beginning of tissular injury. These results suggest that urinary excretion of Oat5 might be an early indicator of mercury-induced nephropathy, which predicts the perturbation before the manifestation of histopathological damages.     

Effect of N-N'-diphenyl-p-phenylenediamine pretreatment on urinary enzyme excretion in cisplatin nephrotoxicity in rats

Two days after cisplatin was injected into rats, urinary N-acetyl-beta-D-glucosaminidase (NAG) and gamma-glutamyltranspeptidase (gamma-GTP) activities increased. The urinary excretion of NAG continued to rise until 4 days after the injection of cisplatin, the last day examined. However, the increase in urinary gamma-GTP excretion which lasted for 2 days returned to its control level 4 days after cisplatin injection. The alkaline phosphatase activity in urine was unaffected by cisplatin injections. The antioxidant N-N'-diphenyl-p-phenylenediamine attenuated these increases in enzyme activities caused by cisplatin. The results of this study suggest that monitoring the change in urinary activities of some enzymes is the method of choice for detecting cisplatin nephrotoxicity and that the increase may involve the generation of free radicals by cisplatin.     

Distal nephrotoxicity of cisplatin demonstrated by urinary kallikrein excretion and morphological study in rats

The nephrotoxic effect of cisplatin (4 mg/kg body wt, i.p. injection) was specifically evaluated on the distal tubule. We measured both the tissue concentration and the urinary excretion of kallikrein (UKE), a serine protease mainly synthesized and secreted in the distal connecting tubular cells. In a parallel morphological study, we evaluated the tissue lesions. On the basis of UKE, the three distinct phases of nephrotoxicity were observed. The induction phase, 1 day after cisplatin injection, was associated with a transient increase in UKE. During the maintenance phase, the kallikrein concentration was significantly decreased both in renal cortex and urine for up to 10 days, suggesting an alteration in the biosynthesis with a decrease in the activation of inactive kallikrein. The recovery phase, 21 days after cisplatin injection, was suggested by the incomplete but significant tendency to return towards control values of active UKE. Histological examinations of cisplatin-treated rats showed early lesions of proximal tubules on day 1. The injuries worsened and tubular necrosis was frequently observed on the following days. Distal tubular changes were less marked but vacuolization and desquamation of epithelial cells and swollen and disrupted mitochondria were demonstrated. This study adds new evidence that UKE is a useful and reliable non-invasive index to assess possible nephrotoxic effects in the distal tubule which are also directly visualized by histological lesions.     

Early onset of cisplatin-induced nephrotoxicity in streptozotocin-diabetic rats treated with insulin

Mechanism of protection against cisplatin nephrotoxicity in streptozotocin-diabetic rats is unclear but is associated with decreased renal platinum accumulation. This study was designed to determine whether normalization of hyperglycaemia by insulin treatment to six week streptozotocin-diabetic rats reversed protection against cisplatin nephrotoxicity. Male Sprague-Dawley rats divided into 3 groups (n=10/group) (1) non-diabetic (2) untreated streptozotocin-diabetic and (3) insulin-treated streptozotocin-diabetic groups were rendered diabetic using streptozotocin (65 mg/kg body weight, intravenous). At the end of 6 weeks, Group 3 animals were treated with insulin (subcutaneously) for 21 days to normalize glucose. After 21 days of insulin treatment, the mean +/- S.D. plasma glucose (mg%) in Group 3 animals at 144.8 +/- 22.03, was significantly lower than Group 2 animals (412 +/- 24.69) and comparable to age-matched non-diabetic (Group 1) animals. Blood urea nitrogen at 24 hr after intraperitoneal administration of cisplatin (5 mg/kg body weight) increased by a factor 2.5 in Group 3 compared to 1.1 and 1.3 in Group 1 and Group 2 animals respectively. In the same animals, at 96 hr the blood area nitrogen increased by a factor of 3.2 and 2.9 in Group 1 and Group 3 respectively compared to 1.14 for Group 2 animals. Renal platinum levels in Group 1, Group 2 and Group 3 after 96 hr after cisplatin administration were 6.92 +/- 0.83, 3.46 +/- 0.77 & 6.20 +/- 0.64 (microg/g wet weight of tissue) respectively. Results indicate that 21 day insulin treatment to streptozotocin-diabetic animal reverses protection against cisplatin toxicity. Moreover, insulin treatment increased the susceptibility of streptozotocin-diabetic rats to cisplatin-induced renal toxicity.     

Increased urinary excretion of carnitine in patients treated with cisplatin

Objective: To study the effect of cisplatin on plasma concentrations and urinary excretion of carnitine in ten patients with different malignancies treated with chemotherapy. Methods: Carnitine concentrations were determined using a radioenzymatic assay and other metabolites by routine methods of clinical chemistry. Renal clearances were calculated by dividing urinary excretions by the respective plasma concentrations. Results: Before treatment, all patients had a normal plasma carnitine concentration. During treatment with cisplatin, the plasma total carnitine concentration increased by approximately 30% and normalized 7 days after stopping therapy. Urinary excretion of total carnitine increased by a factor of 10 during cisplatin administration and also normalized 7 days after cessation of chemotherapy. This increase was due to excretion of both free carnitine and acylcarnitine and averaged approximately 1 mmol carnitine per day. Similarly, urinary clearance of total carnitine was increased during therapy with cisplatin by a factor of approximately 8 and returned to normal 7 days after chemotherapy. In comparison, patients with similar malignancies treated with radiotherapy showed no significant increase in renal carnitine excretion. Similar to urinary excretion of carnitine, excretion of glucose and phosphate, two metabolites also reabsorbed by the proximal tubule of the nephron, was increased during therapy with cisplatin. There was a strong linear correlation between urinary excretion of free carnitine and acylcarnitines. Conclusions: Treatment with cisplatin is associated with a tenfold increase in renal carnitine excretion, most likely due to inhibition of carnitine reabsorption by the proximal tubule of the nephron. Well-nourished patients support this loss of carnitine even after repeated cycles of chemotherapy without developing hypocarnitinaemia. However, cachectic patients with decreased dietary carnitine uptake may develop carnitine deficiency when treated repeatedly with chemotherapies including cisplatin. Received: 26 November 1997 / Accepted in revised form: 25 May 1998     

Compensation increase in organic anion excretion in rats with acute biliary obstruction: role of the renal organic anion transporter 1

The purpose of the present study was to examine in rats the effects of acute bile duct ligation on the expression of the organic anion transporter 1 in the kidney and the consequences of these effects on the systemic clearance of organic anions, particularly on P-aminohippurate (PAH) clearance, since it has been viewed as the prototypic organic anion. Male Wistar rats underwent bile duct ligation (BDL rats). Pair-fed sham-operated rats served as controls. All studies were carried out 21 h after surgery. Our data revealed that BDL rats had a higher expression of organic transporter 1 protein in kidney cortex homogenates. Accordingly, systemic clearance of PAH and urinary excretion of PAH were both higher in BDL rats. These findings suggest that impairment of the liver function after BDL is followed by a distinct and statistically significant increase in renal excretion of PAH, indicating a possible compensation mechanism.     

Increased excretion of urinary 20-HETE in rats with cyclosporine-induced nephrotoxicity

The present study examined the contribution of 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) in cyclosporine A (CsA)-induced renal nephrotoxicity. Treatment of rats with CsA (50 mg/kg) for 9 days induced renal damage as indicated by marked increase in urine flow (from 9.0 +/- 0.3 ml/day to 46.6 +/- 7.1 ml/day) and a 3 - 5-fold rise in blood urea nitrogen (BUN) levels. The urinary excretion of 20-HETE increased from 164 +/- 5 ng/day (N = 5) to 2432 +/- 290 ng/day (N = 5, P<0.01) after 9 days of CsA treatment. The increase in the urinary excretion of 20-HETE in the CsA treated rats was highly correlated with the increase in BUN levels (r = 0.819, P<0.001) and urine volume (r = 0.832, P<0.001). Immunohistochemical examination of kidney revealed that expression of cytochrome P450 4A (CYP4A) protein was markedly enhanced in the proximal tubules of CsA-treated rats. These results indicate that CsA-induced nephrotoxicity in rats is associated with a marked elevation in the renal production of 20-HETE and that 20-HETE may contribute to the pathophysiological condition of CsA-induced nephrotoxicity.     

Effect of buthionine sulphoximine, glutathione and methimazole on the renal disposition of cisplatin and on cisplatin-induced nephrotoxicity in rats: pharmacokinetic-toxicodynamic analysis

The aim of this study was to classify the protective mechanisms of DL-buthionine-(S,-R)-sulphoximine, glutathione and methimazole on cisplatin-induced nephrotoxicity in rats. An Emax model was used to study the effect of these compounds on the pharmacokinetics of cisplatin, especially renal handling and intra-renal biotransformation. Cisplatin (5 mg kg(-1)) was administered as an intravenous bolus to rats treated with either 0.9% NaCl (control), buthionine sulphoximine, glutathione or methimazole. The blood urea nitrogen level was monitored to estimate cisplatin-induced nephrotoxicity. To estimate renal handling of cisplatin, cisplatin was infused intravenously to rats treated with 0.9% NaCl, buthionine sulphoximine, glutathione or methimazole. The concentrations of unchanged cisplatin in plasma, urine and kidney were determined by a post-column derivatization HPLC method. The relationship between the pharmacokinetics and toxicodynamics of cisplatin was analysed using a sigmoid Emax model. All compounds studied ameliorated significantly the nephrotoxicity of cisplatin. The renal accumulation of cisplatin was reduced significantly by pretreatment with buthionine sulphoximine but not by either glutathione or methimazole. Although glutathione treatment did not affect the renal accumulation of cisplatin, it significantly decreased the binding of cisplatin to the intrarenal organelle and the decreased binding was well correlated to the decrease of the blood urea nitrogen level. In summary, pharmacokinetic-toxicodynamic analysis will be useful for classifying the protective mechanism of cisplatin-induced nephrotoxicity.     

Renal accumulation and urinary excretion of cisplatin in diabetic rats.

Previous work has demonstrated that cisplatin nephrotoxicity was attenuated in streptozotocin (STZ)-induced diabetic rats. The following studies investigated the hypothesis that renal cisplatin accumulation was reduced in diabetic rats. Male Fischer 344 (F344) rats were injected with 32 mg/kg STZ (i.p.) or citrate buffer. Renal platinum (Pt) accumulation was quantitated 0-96 h after the administration of 5 mg/kg cisplatin (i.p.) to normoglycemic and diabetic rats (greater than or equal to 4/group). Total renal Pt accumulation was decreased (P less than 0.05) in the diabetic rats, when compared to the normoglycemic group, 6-48 h after cisplatin injection. Further studies were also conducted to examine if urinary cisplatin excretion was enhanced in diabetic relative to normoglycemic groups. Urinary Pt excretion was quantitated 0-96 h following cisplatin (5 mg/kg, i.p.) administration. Pt excretion was increased in the diabetic group relative to the normoglycemics when comparisons were made on the basis of Pt excreted per hour or cumulative Pt excretion. Differences were also detected in urinary Pt concentration. The diabetic group had a lower urinary concentration of the metal 12-96 h after cisplatin injection. These findings suggest that the reduction in nephrotoxicity in diabetic rats may be at least partially due to decreased renal accumulation as well as altered renal excretion.     

Organic anion transporter 2 (SLC22A7) is a facilitative transporter of cGMP

The second messenger, cGMP, mediates a host of cellular responses to various stimuli, resulting in the regulation of many critical physiologic functions. The existence of specific cGMP transporters on the plasma membrane that participate in the regulation of cGMP levels has been suggested in a large number of studies. In this study, we identified a novel plasma membrane transporter for cGMP. In particular, we showed that hOAT2 (SLC22A7), a member of the solute carrier (SLC) superfamily, was a facilitative transporter for cGMP and other guanine nucleotides. hOAT2, which is ubiquitously expressed at high levels in many cell types, was previously thought to primarily transport organic anions. Among purine and pyrimidine nucleobases, nucleosides, and nucleotides, hOAT2 showed the greatest preference for cGMP, which transported cGMP with a K(m) value of 88 +/- 11 muM and exhibited between 50- and 100-fold enhanced uptake over control cells. Our data revealed that hOAT2 is a bidirectional facilitative transporter that can control both intracellular and extracellular levels of cGMP. In addition, we observed that a common alternatively spliced variant of hOAT2 demonstrated a complete loss of transport function as a result of a low expression level on the plasma membrane. We conclude that hOAT2 is a highly efficient, facilitative transporter of cGMP and may be involved in cGMP signaling in many tissues. Our study suggests that hOAT2 represents a potential new drug target for regulating cGMP levels.     

Organic anion transporter 1 (OAT1) involved in renal cell transport of aristolochic acid I

Aristolochic acids (AAs) are a family of structurally related nitrophenanthrene carboxylic acids that are present in medicinal herbs such as Aristolochia species. The organic anion transporters (OATs) of the solute carrier (SLC22) gene family located in the renal proximal tubules play a key role in the excretion of a variety of exogenous and endogenous compounds. However, it is unclear how AAs permeate into renal epithelial cells. In this regard, we investigated the role of rat OAT1 ([rOAT1] SLC22A6) in the cellular uptake of AAI in vitro and in vivo. A concentration- and time-dependent intracellular accumulation of AAI was observed in rOAT1-transfected human embryonic kidney 293 (HEK293) cells, which was 2- to 6-fold higher than the control cells. There was a significantly increased rate of cellular apoptosis in rOAT1-transfected HEK293 cells than control cells after AAI treatment. Para-aminohippuric acid (PAH) significantly reduced the intracellular accumulation of AAI in rOAT1-transfected HEK293 cells. Administration of AAI for 35 days in rats caused significantly reduced expression of OAT1 in basolateral membrane and declined renal clearance of PAH as well as renal proximal tubule injuries. These findings indicate that AAI is taken up by OAT1, which then exert its intracellular toxic effects on renal proximal tubule cells, which in turn damage functional OAT1 and may further disturb the transport of its substrates.     

Pharmacokinetics and toxicodynamics of cisplatin and its metabolites in rats: relationship between renal handling and nephrotoxicity of cisplatin

The renal handling of cisplatin and its metabolites and the relationship between the pharmacokinetics of these platinum species in the kidney and nephrotoxicity in rats were studied by carrying out pharmacokinetic-pharmacodynamic analysis. Rats received cisplatin intravenously as a bolus (2-10 mgkg(-1)) or by constant infusion (55 and 140 microg min(-1) kg(-1)). After intravenous administration of each platinum species, the platinum concentrations of unchanged cisplatin and its mobile and fixed metabolites were determined separately. Nephrotoxicity was estimated by measuring the blood urea nitrogen (BUN) levels and the sigmoid Emax model was used to determine the relationship between pharmacokinetic parameters and BUN levels 5 days after cisplatin administration. Cisplatin and its mobile metabolites in plasma distributed more rapidly and extensively into the kidney (mean apparent kidney-to-plasma concentration ratios were 2.69 and 7.12 mL (g tissue)(-1), respectively) than into the liver (less than 1 mL (g tissue)(-1)). Concomitant administration of mobile metabolites did not significantly alter the disposition of cisplatin. Nephrotoxicity, estimated by measuring BUN levels, appeared to be related to the plasma concentration of intact cisplatin, not total platinum, because mobile metabolites formed from cisplatin showed little nephrotoxicity. The sigmoid Emax model showed the maximum BUN level reached after cisplatin administration was related to the area under the renal cisplatin concentration-time curve (AUCk).     

Triiodothyronine (T3) increases cisplatin nephrotoxicity in young and adult rats

The influence of pretreatment with triiodothyronine (T3) on cisplatin (CP)-induced nephrotoxicity was investigated in 10- and 55-day-old rats. Triiodothyronine pretreatment enhanced CP proteinuria in young and adult rats, and increased blood urea nitrogen concentration in 55-day-old rats. As T3 decreased Pt concentrations in renal tissue, the enhanced nephrotoxicity of CP by T3 must have another mechanism. Enhanced CP nephrotoxicity is discussed in connection with an increase of glutathione concentration obtained in renal tissue as a consequence of T3 pretreatment.     

Phenolic extract of soybean (Glycine max) attenuates cisplatin-induced nephrotoxicity in rats

The present study investigated the modulatory role of phenolic extract of soybean (PESB) in a rat model of nephrotoxic acute renal failure induced by cisplatin. Cisplatin (2 mg/kg/day) was administered to the rats for 5 days and the animals were pretreated with PESB (250–1000 mg/kg). Blood urea nitrogen reduced by 49.8% and 59.0%, serum creatinine by 34.7% and 62.1% and urinary N-acetyl-β-d-glucosaminidase also decreased by 37.7% and 49.2% following treatment with 250- and 500-mg/kg doses of the extract respectively in the cisplatin-treated rats. The extract also significantly increased renal myeloperoxidase activity by 26.8% and 40.6% at these doses. PESB also decreased renal xanthine oxidase activity and serum nitrate/nitrite in the cisplatin-treated rats. In addition, PESB significantly attenuated the marked renal oxidative damage that accompanied cisplatin treatment. The extract improved liver histology and significantly increased the activities of the antioxidant enzymes measured [superoxide dismutase, catalase, glutathione-S-transferase], prevented glutathione depletion and decreased malondialdehyde level following cisplatin treatment. Furthermore, cisplatin-induced decrease in the activities of glucose-6-phosphatase and 5′-nucleotidase in these rats was attenuated only at 250 mg/kg dose of the extract. We concluded therefore that PESB via antioxidant and possibly anti-inflammatory actions offered protective benefit against cisplatin-mediated acute toxic injury to the kidney.     

Organic anion transporter (Slc22a) family members as mediators of toxicity

Exposure of the body to toxic organic anions is unavoidable and occurs from both intentional and unintentional sources. Many hormones, neurotransmitters, and waste products of cellular metabolism, or their metabolites, are organic anions. The same is true for a wide variety of medications, herbicides, pesticides, plant and animal toxins, and industrial chemicals and solvents. Rapid and efficient elimination of these substances is often the body's best defense for limiting both systemic exposure and the duration of their pharmacological or toxicological effects. For organic anions, active transepithelial transport across the renal proximal tubule followed by elimination via the urine is a major pathway in this detoxification process. Accordingly, a large number of organic anion transport proteins belonging to several different gene families have been identified and found to be expressed in the proximal nephron. The function of these transporters, in combination with the high volume of renal blood flow, predisposes the kidney to increased toxic susceptibility. Understanding how the kidney mediates the transport of organic anions is integral to achieving desired therapeutic outcomes in response to drug interactions and chemical exposures, to understanding the progression of some disease states, and to predicting the influence of genetic variation upon these processes. This review will focus on the organic anion transporter (OAT) family and discuss the known members, their mechanisms of action, subcellular localization, and current evidence implicating their function as a determinant of the toxicity of certain endogenous and xenobiotic agents.     

Time course of chronic oral cadmium nephrotoxicity in Wistar rats: excretion of urinary enzymes.

Twelve male and female Wistar rats each received cadmium (as CdCl2) in their diet at concentrations of 0, 10, 50, and 250 ppm for 72 weeks. After 1, 4, 8, 13, 18, 26, 32, 45, 57, and 68 weeks a total of 8 enzymes from different cellular compartments of the nephron were measured. At the end of the study period, the kidneys were examined histopathologically. Concentrations up to and including 50 ppm did not induce any adverse effect. At 250 ppm, growth of male and female animals was markedly retarded. Significantly increased activities of the cytosolic phosphohexose isomerase were excreted by males and females receiving 250 ppm at all timepoints from week 13. The values of the mitochondrial glutamate dehydrogenase were mostly elevated from week 1 to 57, however, due to a wide scatter range, were only occasionally significantly different from control values. The brush border enzymes (gamma-glutamyl transferase, alkaline phosphatase and leucine arylamidase) were not changed in a relevant manner in female rats, while in 250 ppm males the excreted activity of ALP and LAP from week 1 to week 18, and that of GGT during the entire study period were significantly lower than the control values. Excretion of the lysosomal enzymes aryl sulfatase A, beta-galactosidase, and beta-N-acetyl-D-glucosaminidase was at no time influenced in a noteworthy manner. Histopathology after 72 weeks revealed chronic but also acute degenerative changes in the kidneys of 250 ppm males and females. A comparison of published data on persons having undergone high cadmium exposure with the results presented here shows remarkable differences.     

Organic anion transporting polypeptides (OATPs): regulation of expression and function

Eleven members of the human organic anion transporter (OATP) family (grouped into six families) facilitate the Na(+)- independent transmembrane transport of various endo- and xenobiotics (bile acids, bilirubin, steroid hormone conjugates, thyroid hormones, prostaglandins, clinically used drugs, and toxins). OATPs are 12-transmembrane glycoproteins (643-722 amino acids) and contain many conserved structural features, for example, eleven cysteines in the large extracellular loop 5. They are important for proper transport, for which translocation of substrates through a central, positively-charged pore in a rocker-switch-type mechanism has been proposed. Although OATPs are expressed in various cells and tissues, some members show a more restricted pattern (well-studied OATP1B1/OATP1B3 in liver, OATP4C1 in kidney, and OATP6A1 in testis). In cancer, the distribution pattern is no longer maintained, and OATPs, like OATP1B3, become upregulated in malignant tissues (colon, breast, prostate). Studies in cell lines and animal models further revealed that the expression of OATPs is regulated in a cell- and tissue-specific way by cytokines and activation of nuclear receptors (LXR, FXR, PXR, CAR, HNF4). Also epigenetic mechanisms and postranslational modifications influence their expression and function. Therefore, changes in the expression of OATPs under pathological conditions will influence transport processes causing an altered accumulation of OATP substrates in cells of excretory organs (intestine, liver, kidney) and on various blood/organ barriers (such as brain, testis, placenta). For drugs, this may result in increased toxicity and adverse drug reactions. Therefore, it is important to improve the knowledge on the regulation and function of individual OATPs, and to apply it for therapeutic considerations.     

川芎嗪对顺铂肾损伤大鼠肾细胞凋亡及凋亡相关蛋白表达的影响

目的探讨川芎嗪(TMP)对顺铂(DDP)大鼠肾毒性有无保护作用。方法DDP8mg·kg-1ip诱导大鼠肾损伤,于给予DDP2d前大鼠分别ip50,100mg·kg-1·d-1TMP,连续5d,于给药d5收集各组大鼠尿液,测24h尿蛋白含量,并于末次给药后4h处死大鼠,测血清尿素氮(BUN)和肌酐(Cr)含量,采用原位缺口末端标记法检测肾脏细胞凋亡,免疫组化SP法检测肾脏Bax和Bcl2蛋白表达水平。结果50,100mg·kg-1TMP组可降低DDP所致大鼠24h尿蛋白及血清BUN和Cr含量的升高(P<0.01);TMP两剂量组也可明显减少肾脏细胞凋亡率(P<0.01),显著增强肾脏凋亡相关蛋白Bcl2的表达,减少Bax表达,并明显降低Bax/Bcl2比值(P<0.01)。结论TMP可能通过降低凋亡相关蛋白Bax和增强Bcl2的表达,并降低Bax/Bcl2比值而抑制DDP引起的肾细胞凋亡,使肾脏免受损伤的作用。